Investigations into Multiple--Herbicide-Resistant Ambrosia artemisiifolia (Common Ragweed) in Ohio and Glyphosate-Resistance Mechanisms.

摘要

Common ragweed (Ambrosia artemisiifolia) is a weed problem in many places throughout the world. Though it seldom dominates the landscape, common ragweed seems to be able to exploit diverse habitats. Common ragweed is primarily outcrossing and has a high rate of gene polymorphisms, leading to high genetic diversity. This high level of genetic diversity likely plays a major role in the evolution of herbicide-resistant biotypes. Whole-plant bioassays of herbicide dose-response in the greenhouse were used to characterize resistance levels to glyphosate, cloransulam-methyl, and fomesafen herbicides. Additional studies were conducted to provide insight into potential mechanisms that may contribute to the development of resistance to glyphosate in an Ohio ragweed biotype, including 5 enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene sequencing, quantitative PCR of the EPSPS gene, EPSPS enzyme immunoblot and activity/inhibition assays, 31P nuclear magnetic resonance (NMR) studies of glyphosate-treated tissues, and whole-plant absorption and translocation studies using 14C-labeled glyphosate. A single common ragweed population from Clinton County, Ohio exhibited multiple resistance to herbicides at dosages that exceeded the rate required to kill herbicide-sensitive common ragweed biotypes from 4- to 30 fold for glyphosate, < 1000 fold for cloransulam-methyl, and 14- to < 100 fold for fomesafen. This is the first report of a common ragweed biotype with multiple resistance to herbicides from three site-of-action (SOA) groups. Sequencing data indicated the gene coding for EPSPS has a high mutation rate in all studied common ragweed biotypes, but it typically does not code for an altered amino acid sequence in the glyphosate binding area. Additional studies identified alleles of EPSPS coding for proline-to-serine and proline-to-threonine substitutions at amino acid number 106 (based upon the mature maize EPSPS numbering scheme). Previous studies by other authors have found these amino acid substitutions to confer glyphosate resistance in numerous other species. The alleles containing these mutations were not detected in previous studies of Ohio ragweed populations, and it is not known whether these alleles are translated into a functional EPSPS protein. Direct sequence analysis also suggested that there are six-to-eight or more partial- or full-length copies of the EPSPS gene in a typical diploid (2n) common ragweed plant. An immunoblot assay with common ragweed total soluble protein, as well as Palmer amaranth (Amaranthus palmeri) glyphosate-sensitive and EPSPS overexpressing glyphosate-resistant controls, showed a single plant from the glyphosate-resistant biotype with increased EPSPS expression. Quantitative PCR also showed an increased relative EPSPS gene copy number in the same plant. 31P NMR data showed similar uptake of glyphosate into the leaf cells and no vacuolar sequestration in all common ragweed biotypes, with lower sugar-phosphate (including shikimate-3-phosphate) accumulation relative to glyphosate-susceptible common ragweed plants. Similarly, absorption and translocation of 14C-labeled glyphosate over 48 hours did not differ between resistant and susceptible biotypes. More research will be required to unequivocally determine the molecular basis of glyphosate resistance in common ragweed, but accumulated evidence supports the hypothesis that multiple mechanisms of glyphosate resistance are possible within a common ragweed population.